Formation of Supported Tethered Bilayer Lipid Membranes

Au-coated silicon wafers completed with stBLMs^{61 (link)} were made as previously described with minor modifications.^{38 (link)} A mixed self-assembled monolayer (SAM) of the lipid tether compound HC18 (Z20-(Z-octadec-9-enyloxy)-3,6,9,12,15,18,22-heptaoxatetracont-31-ene-1-thiol)^{61 (link)} and β-mercaptoethanol (βME) was obtained by immersing the Au-coated wafers in ethanolic solutions of HC18 and βME (30:70 molar ratio, 0.2 mM final concentration) for >12 h, followed by thorough rinsing with ethanol and drying under N₂. Vesicles (25 mg/mL) of the desired lipid composition were prepared by re-suspending dry lipid films in aqueous buffer (pH 5.5) with 500 mM NaCl. The suspension was sonicated for 1 h in a bath sonicator and extruded at least 30 times through a 100-nm pore size polycarbonate membrane (Whatman, GE Healthcare) using an extrusion kit (Avanti Polar Lipids, Alabaster, AL). Formation of stBLM was achieved by incubating the SAM with vesicles for 1 h followed by rinsing with copious amounts of aqueous buffer containing 100 mM NaCl to remove non-fused vesicles.